| |61 © ARCHIVES OF BUDO | SCIENCE OF MARTIAL ARTS
Judo status is not associated with the a
ngiotensin-converting enzyme insertion/deletion polymorphism
in Jap
anese judo athletes
Toshio Itaka
1ABCD, Kenichiro Agemizu
2BE, Seiji Aruga
3B, Shuichi Machida
1ACDE1 Graduate School of Health and Sports Science, Juntendo University, Chiba, Japan
2 School of Physical Education, Tokai University, Kanagawa, Japan
3 Sports Medical Science Research Institute, Tokai University, Kanagawa, Japan
Received: 14 December 2015; Accepted: 03 February 2016; Published online: 09 March 2016
AoBID: 11004
Abstract
Background & Study Aim: In high-level competitions, judo athletes perform several matches in a day and require a high endurance. The angiotensin-converting enzyme (ACE) polymorphism has been widely studied in relation with endurance and sports status. The purpose of this study was knowledge about the influence of the ACE insertion/deletion (I/D) polymorphism on the judo status and endurance of Japanese athletes.
Material & Methods: This study included 154 Japanese male judo athletes from a top-level university in Japan. They were divided into three groups based on their competitive history (international level, national level, and others). Genomic DNA was extracted from the saliva of each athlete and the distance travelled in a 5-min running test was measured. Genotyping using polymerase chain reaction was performed to detect the ACE I/D polymorphism, rs1799752.
Results: ACE genotype and allele frequency were compared between the judo athletes groups and control subjects. The ACE I/D polymorphism was not associated with judo status in these athletes. Furthermore, no differences were
found among the ACE genotypes pertaining to endurance.
Conclusions: The results of the present study suggest that the ACE gene does not greatly influence the judo status and en-durance in Japanese athletes. However, the ambiguity of these results with studies of other populations judo athletes tend to the conclusion that the precise determination of the biological determinants of talent in judo is a complex phenomenon and requires further study and taking into account the specific genetic methods. Key words: ACE I/D polymorphism, endurance performance, international judo level, national judo level
Copyright: © 2016 the Authors. Published by Archives of Budo Conflict of interest: Authors have declared that no competing interest exists
Ethical approval: The study was approved by the local Ethics Committee Provenance & peer review: Not commissioned; externally peer reviewed
Source of support: JSPS KAKENHI grant numbers 25350783. JSPS KAKENHI grant numbers 24650331. A Tokai University Supporters Association Research and Study Grant.
Author’s address: Shuichi Machida, Graduate School of Health and Sports Science, Juntendo University, 1-1 Hirakagakuendai, Inzai, Chiba 270-1695, Japan; e-mail: machidas@juntendo.ac.jp
Authors’ Contribution: A Study Design
B Data Collection
C Statistical Analysis
D Manuscript Preparation
IntroductIon
The International Judo Federation is a global organization, with 200 affiliated countries. To be successful in international competitions, judo athletes must achieve an excellent level of phys-ical fitness and conditioning. Little suggested that a successful judo performance depends upon the athlete having a high technical and tactical ability, power, strength, endurance and flexibil-ity [1]. Although judo requires explosive power, judo athletes need endurance to exert this power for 5 min or more (until the end of a match). In a high-level competition, judo athletes perform several matches in a day; therefore, judo specifi-cally requires a high endurance.
Aerobic exercises such as running and bike riding can improve the endurance. However, it is known that trainability and training effects are highly individu-alized. Individual differences in endurance are influ-enced not only by environmental but also by genetic factors [2]. To date, at least 206 nuclear genetic markers in the human genome have been found to be associated with physical performance phenotypes and physical fitness [3]. Among these, the
angiotensin-converting enzyme (ACE) gene has been extensively
studied for its association with endurance [4, 5].
ACE insertion/deletion (I/D) polymorphism
was first shown to be a genetic factor that influ-ences human physical performance and trainabil-ity in 1998. In that study, Montgomery et al. [6] reported that the ACE I/D polymorphism influ-enced endurance in elite climbers and response to training in British army recruits. Since then, many studies have demonstrated significant associations between the I allele of the ACE I/D polymor-phism and superior endurance in elite athletes [7 ,8]. In a meta-analysis, Ma et al. [5] found strong evidence for associations between the ACE II gen-otype and endurance events in Caucasian athletes. Some studies [9, 10] have defined endurance-ori-ented sports as those involving more than 5 min of competition, including swimming a distance of 500 m or more and track and field events lasting for at least 5 minutes. Although judo matches can sometimes end in less than 5 min, the designated time for each match is 5 min or more. Therefore, in case of the investigation of the relationship between gene polymorphism and sports status, judo seems to involve the endurance-oriented sports. Cieszczyk et al. [11] reported that the frequency of the I allele in the ACE gene poly-morphism was significantly higher in Polish and
Lithuanian judo athletes than in other Caucasian control subjects. However, there have been no reports of any association between the ACE I/D polymorphism and judo status or endurance abil-ity in Japanese judo athletes. Since the Olympics Games of Tokyo in 1964, the Japanese judo team has won 36 gold medals.
The purpose of this study was knowledge about the influence of the ACE insertion/deletion (I/D) polymorphism on the judo status and endurance of Japanese athletes.
We hypothesized that: (1) the frequency of the I allele in the ACE gene polymorphism may be higher in international-level judo athletes than in other judo athletes or controls; (2) the ACE genotype may be associated with endurance in judo athletes.
MaterIalsandMethods
Participants
The study included 154 Japanese male judo athletes belonging to the judo club of Tokai University, a club that has produced many inter-national-level judo athletes. The athletes were divided into three groups based on their results (sports level of judo) in international- and national-level competitions (Table 1). Sixteen athletes were classified as “international level” (winners of national championships or par-ticipants in international competitions), 36 as “national level” (athletes ranking among the top eight in a college-level competition), and 102 as “others” (members of the University Judo Club). They were also classified into three cumula-tive weight categories: light (60 kg and 66 kg), medium (71 kg, 73 kg, 81 kg, and 90 kg), and heavy (100 kg and over 100 kg). The controls were individuals of known genotype from the general Japanese population, as reported in pre-vious studies: ACE-Controls, n = 5,679 [12-21]. The study was approved by the Ethics Committee of Tokai University in Japan and was con-ducted according to the Declaration of Helsinki. The objectives and methods of the study were explained to the subjects, and written informed consent was obtained from each of them.
Genotyping
DNA was extracted from the saliva of all subjects using the QIAamp DNA Mini Kit (Qiagen, Italy) Allele – one of the alternative
versions of a gene at a given location (locus) along a chromosome.
Endurance – noun the ability or power to bear prolonged exertion, pain or hardship endurance athlete.
according to the manufacturer’s protocol. The
ACE I/D (rs1799752) polymorphism was
deter-mined using polymerase chain reaction (PCR), and the resulting PCR products were genotyped using agarose gel electrophoresis (Mupid-2plus, a submarine type electrophoresis system, Advance, Japan). The primers (Fasmac Co. Ltd., Japan) used for the ACE I/D polymorphism were as fol-lows: forward
5′-GCCCTGCAGGTGTCTGCAGCATGT-3′ and reverse
5′-GGATGGCTCTCCCCGCCTTGTCTC-3′, generating a fragment of 597 base pairs (bp) [22]. DNA amplification was performed using a ther-mal cycler Program Temp Control System PC-816 (Astec Co. Ltd., Japan). The PCR con-ditions were as follows: initial denaturation for 2 min 30 s at 94.0°C; 35 cycles of denaturation for 30 s at 94.0°C, annealing for 45 s at 56.0°C, syn-thesis for 2 min at 72.0°C, and final extension for 10 min at 72.0°C. Homozygotes produced either a single 597 bp band (II) or a 319 bp band (DD); heterozygotes (ID) produced both bands. All sub-jects were categorized according to whether they exhibited II, ID, or DD genotypes.
To avoid the misclassification of ID heterozy-gotes as DD homozyheterozy-gotes, a second PCR was performed for the ID and DD genotype samples. The insertion-specific primers used for the ACE I band polymorphism were as follows: forward 5′-TGGGACCACAGCGCCCGCCACTAC-3′ and reverse
5′-TCGCCAGCCCTCCCATGCCCATAA-3′, generating a fragment of 335 bp [22].
The PCR conditions were as follows: initial turation for 1 min at 94.0°C, 35 cycles of dena-turation for 30 s at 94.0°C, annealing for 45 s at 67.0°C, and synthesis for 2 min at 72.0°C. The reaction yielded a 335 bp amplicon only in the presence of the I allele, whereas no product was generated in samples that were homozygous for DD. The digested products were separated using 2% agarose (Agarose for 150–1,500 bp fragments, Nacalai Tesque Inc., Japan) gel electrophoresis, stained with ethidium bromide, and visualized under ultraviolet light. All genotyping analyses were conducted blind to the subjects’ identities.
The 5-min running test
Considering the ideal duration of a judo match, a 5-min running test was conducted with each athlete as an index of their endurance ability. We measured the total distance traveled in the 5-min running [23]. This test involved three trials at 5 min intervals, and was conducted in a 400 m ath-letic field in the morning.
Statistical analysis
The genotype distribution was evaluated for con-formity with the Hardy–Weinberg equilibrium using a chi-square test with two degrees of free-dom. The genotype distribution and allele fre-quency were compared between the groups of judo athletes and ACE-controls [12-21]. using the chi-square test. Analysis of variance (ANOVA) was used to compare the performance of the dif-ferent genotype groups in each test. The level of significance was set at p<0.05, and all the statis-tical analyses were conducted using the Statisstatis-tical Package for the Ekuseru-Toukei 2012 (Social Survey Research Information Co. Ltd., Japan).
Table 1. Characteristic of judo male athletes (n = 154).
Sports level of judo N
Cumulative weight categories
Light
(-60, -66kg)
Middle
(-71*, -73, -81, -90kg)
Heavy
(-100, +100kg)
International 16 3 7 6
National 36 9 14 13
Others 102 22 54 26
All judo athletes 154 34 75 45
results
The genotype frequencies for the ACE I/D polymorphism (II = 36.4%, ID = 46.1%, DD = 17.5%) were as per the Hardy–Weinberg equi-librium (p>0.05). The ACE genotype frequencies of the ACE-control subjects have been previously published. The frequencies of the ACE I/D poly-morphism in the controls were 41.1% for II, 46.1% for ID, and 12.8% for DD. There was no significant difference between each group and the control subjects in the ACE genotype and allele frequency (Table 2).
There was no significant difference between each group and the control subjects in the ACE geno-type and allele frequency (Table 3). However, the higher frequency of the D allele in the heavy cat-egories group (45.6%) compared with the ACE-controls (35.8%) was close to being statistically significant (p = 0.06).
Of the 154 judo athletes, 75 undertook the 5-min running test. These included 28 with II, 30 with ID, and 17 with DD genotypes (with 2 international-level, 23 national-international-level, and 50 other athletes). One-way ANOVA indicated no significant differ-ences between the genotype groups. Moreover, one-way ANOVA indicated no significant differ-ences between the genotype groups in the total distance covered or decrease in distance between the first and third trials (Figure 1). There were no significant differences in the distance travelled in the 5-min running of first trials among gen-otype groups.
dIscussIon
In this study, we found no association between the ACE I/D polymorphism and judo status or endurance in Japanese athletes. ACE is a key enzyme in the renin–angiotensin system (RAS) that regulates blood pressure via the production of angiotensin II and converts angiotensin I to angiotensin II. An I/D polymorphism in the ACE gene has been found to have a strong effect on circulatory and tissue ACE activity [24, 25]. The
ACE gene, located on chromosome 17 comprises
a 287 bp I/D polymorphism in intron 16, which results in three genotypes (II, ID, and DD) [26]. Homozygotes for the I allele (II) have significantly lower ACE activity than heterozygotes (ID), and heterozygotes have lower ACE activity than homozygotes (DD) [24]. Low ACE activity (the II genotype) results in vasodilatation. Therefore, it is believed to enhance endurance performance, and the ACE II genotype has been found to be predominant in elite athletes that require a high endurance, including long-distance runners [4, 7], long-distance swimmers [4], and ironman triath-letes [27]. Therefore, we hypothesized that the
ACE II genotype was associated with endurance
ability in judo athletes. However, in this study, no differences were found among the ACE genotypes pertaining to endurance in Japanese judo athletes. A meta-analysis study provides evidence for the strong association between the ACE II genotype and endurance events in Caucasian [5]. However, previous studies have reported ethnic differ-ences of phenotype (endurance) in the role of the
ACE I/D polymorphism between Caucasians and
Table 2. The genotype andallele frequency of the ACE I/D polymorphism in the different judo status groups.
Level of judo N Genotype frequency n (%) p-value Allele frequency n (%) p-value
II ID DD I allele D allele
All judo athletes 154 56(36.4) 71(46.1) 27(17.5) 0.18 183(59.4) 125(40.6) 0.09
International
+ national 52 18(34.6) 26(50.0) 8(15.4) 0.61 62(59.6) 42(40.4) 0.33
International 16 7(43.8) 5(31.3) 4(25.0) 0.27 19(59.4) 13(40.6) 0.57
National 36 11(30.6) 21(58.3) 4(11.1) 0.33 43(59.7) 29(40.3) 0.43
Others 102 38(37.3) 45(44.1) 19(18.6) 0.22 121(59.3) 83(40.7) 0.15
Comparison with controls was by chi-square test. Control data were made by the published study in cited reference [12-21].
-Asians [28, 29]. Tobina et al. [29] did not find any association between the ACE II genotype and endurance performance in Japanese elite run-ners. Several genetic polymorphisms have been reported to be predictive of endurance perfor-mance in elite athletes [3]. In other words, our results suggest that ACE polymorphism is not a genetic factor affecting endurance performance in judo athletes.
Only one previous study conducted in Poland and Lithuania reported that the ACE gene was associ-ated with judo status [11]. On the contrary, our
results indicated that ACE polymorphism was not associated with judo status. Cieszczyk et al. [11] found that elite Polish and Lithuanian male judo athletes showed a higher frequency of the I allele of the ACE gene than Caucasian controls (60.7% vs. 44.3%), suggesting that the I allele may be an advantage for performance in judo. In contrast, our data showed that there was no significant dif-ference in the frequency of the ACE I/D polymor-phism in Japanese controls compared with that of the all judo athletes. I allele frequencies of the
ACE gene in Japanese control and all judo
ath-letes were 64.2% and 59.4%, respectively. The
Table 3. The genotype and allele frequency of the ACE I/D polymorphism in the different weight class groups.
Cumulative weight categories N
Genotype frequency n (%)
p-value Allele frequency n (%) p-value
II ID DD I allele D allele
Light
(-60, -66kg) 34 (32.4)11 (44.1)15 (23.5)8 0.16 (54.4)37 (45.6)31 0.09
Middle (-71*, -73, -81,
-90kg) 75
31
(41.3) (45.3)34 (13.3)10 0.99 (64.0)96 (36.0)54 0.96
Heavy (-100,
+100kg) 45 (28.9)13 (51.1)23 (20.0)9 0.16 (54.4)49 (45.6)41 0.06
Comparison with controls was by chi-square test. Control data were made by the published study in cited reference [12-21]
ACE-Controls 5,679 (41.1)2,336 2,617(46.1) (12.8)726 (64.2)7,289 (35.8)4,069
*71 kg (according to the old regulations of sport judo)
frequency of the I allele in Polish and Lithuania judo athletes (60.7%) was similar to that in the Japanese controls (64.2%) and Japanese judo athletes (59.4%). Therefore, ethnic differences between Japanese and Caucasians controls in the ACE I/D polymorphism may account for the discrepancy between the two results. Hence, the
ACE I/D polymorphism could be used to
pre-dict judo status in Polish and Lithuanian, but not Japanese, judo athletes.
There was a near-significant result for the higher frequency of the D allele in the heavy weight cat-egories athletes compared with the ACE-controls. To date, no study has investigated the effect of genetic polymorphisms on weight categories in sports. It is possible that the gene polymorphism may have an influence on the weight class of judo, but the mechanism responsible for this phenom-enon of the ACE I/D polymorphism is unknown. Further studies are required to examine the asso-ciation between ACE I/D polymorphism and body mass in athletes.
The present study had some limitations. First, the results of two of our judo status groups (interna-tional and na(interna-tional level) were probably limited by the relatively small sample size of each group and low statistical power. A sufficient sample size of international-level athletes would be necessary
to attain robust conclusions regarding the asso-ciation between genetics and sports performance. However, although the sample size of the inter-national judo athletes in the study was small, 16 athletes in this group were winners of National Championships and the group included Olympic champions and winners of World Championships. Second, we were unable to measure general endurance in terms of maximum oxygen uptake.
conclusIons
The findings of the present study suggest that the ACE I/D polymorphism was not significantly associated with judo status and endurance in Japanese judo athletes. In conclusion, the ACE
I/D polymorphism does not greatly influence the
performance of Japanese judo athletes. However, the ambiguity of these results with studies of other populations judo athletes tend to the con-clusion that the precise determination of the bio-logical determinants of talent in judo is a complex phenomenon and requires further study and tak-ing into account the specific genetic methods.
acknowledgeMent
We wish to thank Ms. Ai Taketomi for her valu-able help. We also thank all volunteers for their participation in this study.
references
1. Little NG. Physical performance attributes of junior and senior women, juvenile, junior, and senior men judokas. J Sports Med Phys Fitness 1991; 31: 510-520
2. Bouchard C, Sarzynski MA, Rice TK et al. Genomic predictors of the maximal O₂ uptake response to standardized exercise training pro-grams. J Appl Physiol (1985) 2011; 110(5): 1160-1170
3. Bray MS, Hagberg JM, Pérusse L et al. The human gene map for performance and health-related fitness phenotypes: the 2006-2007 update. Med Sci Sports Exerc 2009; 41: 35-73 4. Tsianos G, Sanders J, Dhamrait S et al. The ACE
gene insertion/deletion polymorphism and elite endurance swimming. Eur J Appl Physiol 2004; 92: 360-362
5. Ma F, Yang Y, Li X et al. The association of sport performance with ACE and ACTN3 genetic poly-morphisms: a systematic review and meta-anal-ysis. PLos One 2013; 8: e54685
6. Montgomery HE, Marshall R, Hemingway H et
al. Human gene for physical performance. Nature 1998; 393: 221-222
7. Myerson S, Hemingway H, Budget R et al. Human angiotensin I-converting enzyme gene and endurance performance. J Appl Physiol 1999; 87: 1313-1316
8. Alvarez R, Terrados N, Ortolano R et al. Genetic variation in the renin-angiotensin system and athletic performance. Eur J Appl Physiol 2000; 82: 117-120
9. Ginevičienė V, Pranculis A, Jakaitienė A et al. Genetic variation of the human ACE and ACTN3 genes and their association with functional muscle properties in Lithuanian elite athletes. Medicina (Kaunas) 2011; 47: 284-290
10. Wang G, Mikami E, Chiu LL et al. Association analysis of ACE and ACTN3 in elite Caucasian and East Asian swimmers. Med Sci Sports Exerc 2013; 45: 892-900
11. Cieszczyk P, Maciejewska A, Sawczuk M et al. The angiotensin converting enzyme gene I/D polymorphism in elite polish and lithuanian judo
players. Biol Sport 2010; 27: 119-122 12. Sugiyama T, Morita H, Kato N et al. Lack of
sex-specific effects on the association between angiotensin-converting enzyme gene polymor-phism and hypertension in Japanese. Hypertens Res 1999; 22: 55-59
13. Higaki J, Baba S, Katsuya T et al. Deletion allele of angiotensin-converting enzyme gene increases risk of essential hypertension in Japanese men: the Suita Study. Circulation 2000; 101: 2060-2065 14. Zaman MM, Yoshiike N, Date C et al. Angiotensin
converting enzyme genetic polymorphism is not associated with hypertension in a cross-sectional sample of a Japanese population: the Shibata Study. J Hypertens 2001; 19: 47-53
15. Todoroki M, Minami J, Ishimitsu T et al. Relation between the angiotensin-converting enzyme inser-tion/deletion polymorphism and blood pressure in Japanese male subjects. J Hum Hypertens 2003; 17: 713-718
autonomic nervous system function in young healthy Japanese subjects. J Clin Endocrinol Metab 2006; 91: 4676-4681
17. Zhang L, Miyaki K, Araki J et al. Interaction of angiotensin I-converting enzyme insertion-dele-tion polymorphism and daily salt intake influ-ences hypertension in Japanese men. Hypertens Res 2006; 29: 751-758
18. Yamagishi K, Tanigawa T, Cui R et al. High sodium intake strengthens the association of ACE I/D polymorphism with blood pressure in a community. Am J Hypertens 2007; 20: 751-757 19. Tanabe Y, Kawasaki R, Wang JJ et al. Angiotensin-converting enzyme gene and retinal arteriolar nar-rowing: the Funagata Study. J Hum Hypertens 2009; 23: 788-793
20. Yoshihara A, Tobina T, Yamaga T et al. Physical function is weakly associated with angiotensin-converting enzyme gene I/D polymorphism in elderly Japanese subjects. Gerontology 2009; 55: 387-392
21. Okamoto N, Nakatani T, Okamoto Y et al. Increasing the number of steps walked each day improves physical fitness in Japanese commu-nity-dwelling adults. Int J Sports Med 2010; 31: 277-282
22. Lindpaintner K, Pfeffer MA, Kreutz R et al. A prospective evaluation of an angiotensin-con-verting-enzyme gene polymorphism and the risk of ischemic heart disease. N Engl J Med 1995; 332: 706-711
23. Yoshizawa S, Ishizaki T, Honda H. The studies on the development of aerobic work capacity of Japanese rural children and adolescents aged from 4 to 18 years. 1983; 28: 199-214 [in Japanese] 24. Rigat B, Hubert C, Alhenc-Gelas F et al. An
insertion/deletion polymorphism in the angio-tensin I-converting enzyme gene accounting for half the variance of serum enzyme levels. J Clin Invest 1990; 86: 1343-1346
25. Danser AH, Schalekamp MA, Bax WA et al. Angiotensin-converting enzyme in the human
heart. Effect of the deletion/insertion polymor-phism. Circulation 1995; 92: 1387-1388 26. Tiret L, Rigat B, Visvikis S et al. Evidence, from
combined segregation and linkage analysis, that a variant of the angiotensin I-converting enzyme (ACE) gene controls plasma ACE levels. Am J Hum Genet 1992; 51: 197-205
27. Collins M, Xenophontos SL, Cariolou MA et al. The ACE gene and endurance performance dur-ing the South African Ironman Triathlons. Med Sci Sports Exerc 2004; 36: 1314-1320 28. Amir O, Amir R, Yamin C et al. The ACE
dele-tion allele is associated with Israeli elite endur-ance athletes. Exp Physiol 2007; 92: 881-886 29. Tobina T, Michishita R, Yamasawa F et al.
Association between the angiotensin I-converting enzyme gene insertion/deletion polymorphism and endurance running speed in Japanese run-ners. J Physiol Sci 2010; 60: 325-330
